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Toward an Optimized Workflow for Middle-Down Proteomics
Open accessAbstract
Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.
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Metadata
- Authors
- Cristobal, A., Marino, F., Post, H., van den Toorn, H. W. P., Mohammed, S., & Heck, A. J. R.
- Affiliations
- Utrecht University (NL)
- Utrecht University , , Padualaan 8 , ,
- Corresponding author
- Mohammed, Shabaz
- Heck, Albert J. R.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-02-24
- Volume
- 89
- Issue
- 6
- Pages
- 3318-3325
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5362747
- OpenAlex
- W2590064181
- Cited by
- 130
- References
- 62
- Citations by year
- 2026: 7 · 2025: 17 · 2024: 8 · 2023: 20 · 2022: 8 · 2021: 17 · 2020: 18 · 2019: 17 · 2018: 11 · 2017: 5
- FWCI
- 6.88
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Analytical Chemistry (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
- Funders
- Horizon 2020 Framework Programme (686547)
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (184.032.201, 700.10.429, 718.015.003)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- middle-down proteomics, reversed-phase liquid chromatography, electron transfer dissociation, post-translational modification, sequencing depth
- MeSH terms
- HeLa Cells, Humans, Particle Size, Peptide Hydrolases, Peptides, Mass Spectrometry, Proteomics
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